GABAB receptors do not internalize after baclofen treatment, possibly due to a lack of β-arrestin association: Study with a real-time visualizing assay

Authors

  • Yuka Sudo,

    1. Department of Cellular and Molecular Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan
    2. Division of Cancer Pathophysiology, National Cancer Center Research Institute, Tokyo 104-0045, Japan
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  • Minoru Hojo,

    1. Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501, Japan
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  • Yuko Ando,

    1. Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501, Japan
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  • Masafumi Takada,

    1. Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501, Japan
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  • Hiroaki Murata,

    1. Department of Anesthesiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8501, Japan
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  • Shinji Kurata,

    1. Department of Clinical Physiology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8588, Japan
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  • Noriyuki Nishida,

    1. Department of Cellular and Molecular Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan
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  • Yasuhito Uezono

    Corresponding author
    1. Department of Cellular and Molecular Biology, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki 852-8523, Japan
    2. Division of Cancer Pathophysiology, National Cancer Center Research Institute, Tokyo 104-0045, Japan
    • Division of Cancer Pathophysiology, National Cancer Center Research Institute, Tokyo 104-0045, Japan
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Abstract

The mechanism of agonist-induced GABAB receptor (GABABR) internalization is not well understood. To investigate this process, we focused on the interaction of GABABR with β-arrestins, which are key proteins in the internalization of most of the G protein-coupled receptors, and the agonist-induced GABABR internalization and the interaction of GABABR with β-arrestin1 and β-arrestin2 were investigated in real time using GABABR and β-arrestins both of which were fluorescent protein-tagged. We then compared these profiles with those of μ-opioid receptors (μOR), well-studied receptors that associate and cointernalize with β-arrestins. When stimulated by the specific GABABR agonist baclofen, GABABR composed of GABAB1aR (GB1aR) and fluorescent protein-tagged GABAB2R-Venus (GB2R-V) formed functional GABABR; they elicited G protein-activated inwardly rectifying potassium channels as well as nontagged GABABR. In cells coexpressing GB1aR, GB2R-V, and β-arrestin1-Cerulean (βarr1-C) or β-arrestin2-Cerulean (βarr2-C), real-time imaging studies showed that baclofen treatment neither internalized GB2R-V nor mobilized βarr1-C or βarr2-C to the cell surface. This happened regardless of the presence of G protein-coupled receptor kinase 4 (GRK4), which forms a complex with GABABR and causes GABABR desensitization. On the other hand, in cells coexpressing μOR-Venus, GRK2, and βarr1-C or βarr2-C, the μOR molecule formed μOR/βarr1 or μOR/βarr2 complexes on the cell surface, which were then internalized into the cytoplasm in a time-dependent manner. Fluorescence resonance energy transfer assay also indicated scarce association of GB2R-V and β-arrestins-C with or without the stimulation of baclofen, while robust association of μOR-V with β-arrestins-C was detected after μOR activation. These findings suggest that GABABRs failure to undergo agonist-induced internalization results in part from its failure to interact with β-arrestins. Synapse 66:759–769, 2012.© 2012 Wiley Periodicals, Inc.

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